The Regulation of Lymphatic Muscle Cell Contractile Activity by Intracellular Calcium Signals

Abstract

Lymphedema is characterized by chronic edema due to lymphatic insufficiency. Normally, the spontaneous contractions of the collecting lymphatic vessels (cLVs) function to pump lymph against a pressure gradient back to the blood stream, however cLVs from lymphedema patients often display weak or irregular pumping activity. We have recently shown that that mouse lymphatic muscle cells (LMCs) exhibit a diastolic depolarization that sets contraction frequency and is the basis of cLV pacemaking and autorhythmicity. In murine cLVs, this diastolic depolarization is pressure‐dependent and is mediated by the Ca2+ activated chloride channel Anoctamin1 (Ano1). Ano1 is presumed to be activated by the spontaneous Ca2+ transients originating from the sarcoplasmic reticulum (SR), however the identity of the SR Ca2+ channels underlying these diastolic Ca2+ transients has not been rigorously tested using genetic models. We tested the hypothesis that intracellular Ca2+ signals released from the SR are critical in regulating lymphatic pacemaking and contractile activity. We sorted murine LMCs and observed expression of both Itpr1 and RyR2. We tested the role of Itpr1 and RyR2 in the electrical and contractile regulation of the murine inguinal‐axillary cLV (Ing‐Ax) through the use of inducible deletion models (MYH11CreERT2) or mice harboring knock‐in gain of function (GOF) of loss of function (LOF) mutations. These mouse models include Ano1fl/fl, Itpr1fl/fl, Itpr1DK (GOF mutant), Itpr1DA (LOF mutant), RyR2fl/fl, and RyR2VF (GOF mutant). We performed ex vivo isobaric vessel myography in response to a physiological pressure range to determine Ing‐Ax contraction frequency (Freq), contraction amplitude (Amp), and vessel tone (Tone). In some experiments, we also recorded membrane potential using “sharp electrodes” and imaged Ca2+ signals using the genetically encoded cytosolic Ca2+ sensor GCaMP6f or GCaMP8 from pressurized Ing‐Ax cLVs. Ing‐Ax cLVs isolated from the heterozygous Itpr1DA/WT, the homozygous Itpr1DA/DA, and Itpr1smKO mice had significantly reduced contraction Freq and Tone, but also observed significantly higher Amp. Strikingly, Itpr1smKO Ing‐Ax cLVs also lacked the pressure‐dependent chronotropy similar to cLVs from Ano1smKO mice, however Itpr1smKO vessels had unstable periodicity in the contraction rhythm. Ing‐Ax cLVs from RyR2smKO mice had normal contraction Freq, Amp and Tone. In contrast, Ing‐Ax cLVs from the GOF RyR2VF/KO mice had significantly higher Tone and Freq, but significantly reduced Amp. Ing‐Ax cLVs from GOF Iptr1DK mice contracted into a paralytic rigor during the vessel warming and equilibration period likely due to Ca2+overload. These findings suggest that the pressure‐dependent Freq in murine cLVs is mediated by the activation of Ano1 by SR Ca2+ release through Itpr1 and an acceleration of the diastolic depolarization.Support or Funding InformationR01‐H122578 (MD), R01‐HL133256 & R01‐HL137745 (JJ)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.